Author response:
The following is the authors’ response to the original reviews.
eLife Assessment:
The study provides an important advance towards understanding how spatial and temporal transcriptional programs are integrated to regulate lineage-specific chromatin and enhancer activation. The functional evidence is currently incomplete, but the current data provide a solid correlative and conceptual foundation. Functional experiments directly linking Gsb occupancy to chromatin state and regulation of some lineage-specific targets would further strengthen the causal interpretation of the model. Clarifying the scope of conclusions and explicitly acknowledging the technical limitations of current chromatin assays would provide a more balanced interpretation of the manuscript.
We thank the reviewers and editors for their comments on our manuscript. We address here the concerns raised by them.
Public Reviews:
Reviewer #1 (Public review):
Summary:
It has long been known that Drosophila embryonic ventral nerve cord neuroblasts incorporate both spatial and temporal transcription factor expression to generate 30 distinct neuroblasts and lineages per hemisegment. This manuscript aims to elucidate the mechanism by which this integration of spatial and temporal transcription factors occurs through "direct regulation" or "epigenetic regulation". Direct regulation is defined as both spatial and temporal factors binding to open chromatin and working together to dictate specific lineages. Epigenetic regulation is defined as a spatial factor priming the chromatin in a neuroblast-specific manner to allow for the integration of temporal factors to generate specific lineages. The authors conclude that there is a two-step model in which a spatial transcription factor code "primes" the chromatin in terms of accessibility and then recruits temporal factors to ensure lineage-specific enhancer activation.
We thank the reviewer for this clear and succinct summary and for accurately capturing the central idea of the model we propose. In particular, we appreciate that the reviewer highlights the distinction between the previously proposed “direct regulation” and “epigenetic regulation” models, which our work suggests may operate together within neuroblast lineages through a combinatorial spatial transcription factor code — a model we refer to as the “distributed STF code model”.
Strengths:
The authors tested two models, "direct regulation" vs "epigenetic regulation" in a well-defined pool of neural stem cells during normal development.
We thank the reviewer for recognizing this aspect of the study.
Weaknesses:
The data in this study cannot clearly substantiate these two models.
Overall, there are a number of issues that are inconsistent and not supportive of the model proposed in this manuscript. Firstly, there is no evidence of pioneer factor activity in any of the NB lineages described - i.e., any changes in chromatin accessibility being shown over time. The authors must show chromatin conformation changes during the window of spatial transcription factor expression in order to convince the readers of this phenomenon.
Thank you for raising this point. We agree that a developmental time course of chromatin accessibility would be very informative. The difficulty is that, in this system, the relevant sequence unfolds extremely rapidly and across two cellular contexts. Spatial transcription factors such as Gsb are expressed in the neuroectoderm; NBs then delaminate, and Hb expression begins almost immediately — on the order of minutes to tens of minutes. Before delamination there is no NB that can be targeted with lineage-specific drivers, whereas after NB formation the temporal program is already under way. This transition therefore cannot currently be resolved using lineage-restricted Dam profiling.
In most studies, however, pioneer or chromatin-priming activity is inferred from a transcription factor’s ability to bind regions of relatively low accessibility and alter accessibility in a heterologous cellular context, rather than from direct developmental timecourse measurements. Our study provides both types of evidence: Gsb occupies accessible and relatively less-accessible regions in NB5-6 (Figure 4), while ectopic Gsb expression in NB7-4 causes bidirectional chromatin remodelling, with corresponding changes in Hb occupancy (Figure 6 and Figure 6—figure supplement 2).
In light of this technical constraint, and in response to the reviewer’s broader concern, we have revisited our original interpretation. We now emphasise Gsb’s ability to alter chromatin accessibility bidirectionally and use the term “chromatin priming” rather than “pioneering.” We also make clear that Gsb does not act alone to establish the NB5-6 chromatin landscape, but functions within a broader, distributed STF code.
Some examples of relevant manuscript changes are:
- “We found, however, that Gsb’s role in chromatin remodelling was more complex: ectopic Gsb remodelled chromatin bidirectionally with regions both losing and gaining accessibility upon Gsb misexpression.”
- “Thus, Gsb’s chromatin-remodelling capacity is not equivalent to autonomous opening of lineage-appropriate enhancers. Rather, these data support a model in which Gsb contributes to chromatin priming as part of a broader NB-specific STF code…”
- “Thus, Gsb can engage loci and directly restrict chromatin accessibility at bound sites, but its ability to promote accessibility and Hb recruitment likely requires additional NB5-6-specific inputs.”
Secondly, the phenotypic data do not align with the sequencing data - the story would be more cohesive if the sequencing data and phenotypic data were in the same NB subtypes. On one hand, we are shown that Gsb misexpression induces loss of chromatin accessibility in NB 7-4, however in the widespread loss model, we are not shown a phenotype in these NB7-4 - which suggest that the chromatin accessibility at these sites (sites that have already been distinguished as SoIs for that NB subtype) does not play an important role in distinguishing NB 7-4 identity. However, the authors report loss of NB3-5 identity but have no evidence as to how the chromatin has changed (or if it has at all) in that subtype, leaving the readers to wonder how the loss of identity occurred
Thank you for raising this point regarding the alignment between the chromatin and phenotypic analyses. The reviewer’s comment made us realise that the rationale for these experiments may not have been sufficiently clear in the original manuscript and could therefore be perceived as misaligned. We therefore explain the logic of the experimental design here and have edited the manuscript to clarify this point for readers.
The chromatin experiments were designed to test whether Gsb is capable of remodelling chromatin when introduced outside its endogenous domain. NB7-4 provided a convenient heterologous context with clean genetic access for TaDa/CATaDa profiling.
Importantly, although NB7-4 was selected primarily for this technical reason, Gsb misexpression did affect NB7-4’s identity readout: at higher induction levels, the NB7-4-specific Gal4 reporter was lost. Because this same driver was required for lineage-restricted chromatin profiling, we titrated Gsb misexpression to a lower level that preserved reporter activity while still allowing us to assay chromatin accessibility and Hb occupancy. This calibration is documented in the new Figure 6—figure supplement 1. Thus, the calibrated profiling condition should not be interpreted as evidence that Gsb has no effect on NB7-4 identity; rather, it was necessary to retain genetic access to the lineage.
The phenotypic experiments addressed a different question: the developmental requirement for Gsb within its endogenous spatial domain. Our analysis was therefore centred on NB5-6. Gsb depletion caused loss of the NB5-6-specific Lbe reporter and the NB5-6 neuronal markers Ap and Eya, whereas Gsb misexpression produced a partial gain of these markers (Figures 7 and 8). The accompanying changes in row-3/NB3-5 markers emerged as an additional reciprocal phenotype, consistent with Gsb established role in specifying row-5/6 identity while repressing row-3 fates, rather than as an independent endpoint that we had selected.
Thus, the chromatin and phenotypic experiments are complementary: the NB7-4 experiment tests Gsb’s capacity to remodel chromatin in a convenient heterologous context, whereas the NB5-6 experiments establish its developmental function within its endogenous domain. We have revised the manuscript to make this distinction explicit.
Relevant edits made to manuscript:
“Having shown that ectopic Gsb remodels chromatin and alters Hb occupancy in the NB7-4 lineage, we next asked whether altering Gsb function changes NB lineage identity. The misexpression experiments described above had already shown that, when induced at high levels, Gsb misexpression turned off the NB7-4-lineage Gal4 reporter, consistent with Gsb suppressing NB7-4 identity at sufficient levels. In the chromatin-profiling experiments above, this was addressed by inducing lower Gsb misexpression levels that preserved Gal4 expression. Together, these observations suggested that Gsb misexpression can influence lineage identity as well as chromatin state. However, since NB7-4 is not an endogenous Gsb-expressing context, we turned to NB5-6 to test the developmental consequences of altering Gsb function.”
Reviewer #2 (Public review):
Summary:
This article by Bhattacharya et al. investigates how neural stem cells (NSCs, NBs) in Drosophila integrate spatial and temporal cues to activate neuron-specific terminal selector (TS) genes. Prior to this work, it was understood that NSCs utilize spatial transcription factors (STFs) and temporal transcription factors (TTFs) to determine lineage identity and birth order, but the mechanisms of integration were not fully elucidated. The authors employed chromatin profiling techniques to analyze the binding of STFs and TTFs in two specific neuroblast lineages, NB5-6 and NB7-4. They found that Gsb (an STF) binds both accessible and less-accessible chromatin in NB5-6, while En (another STF) binds only to pre-accessible chromatin in NB7-4. The findings support an "STF code" where the combination of pioneer and non-pioneer spatial factors, along with temporal factors, triggers neuroblast-specific enhancer activation and determines lineage identity.
We appreciate the reviewer’s careful summary of our findings and their clear articulation of the STF-code framework that emerges from the work.
Strengths:
The experiments are well-executed, the interpretations are generally sound, and the figures are clear and elegant. However, some conclusions are drawn too broadly without essential functional data. Therefore, additional work is needed to more effectively convey the central message.
We thank the reviewer for their positive assessment of the experiments, interpretation, and figures, and we respond to their specific concerns below.
Weaknesses:
(1) Integration of TaDa and functional data on Gsb for the STF model
The authors demonstrate that TaDa profiling maps Gsb binding across the genome and identifies candidate chromatin-priming sites in NB5-6. Gsb LOF/GOF experiments reveal effects on NB identity. Combining TaDa data with LOF and GOF analyses indicates that Gsb influences NB5-6 specification by binding to both open and relatively closed chromatin, helping maintain NB5-6 identity while limiting NB3-5 fate.
However, the study does not establish a direct link between specific LOF/GOF phenotypes and particular genomic targets. For instance, analyzing Gsb occupancy at lineage-specific identity factors or terminal selector genes (such as Lbe, Ap, or Eya for NB5-6; and Ems, etc., for NB3-5) in wild-type and manipulated conditions (Gsb misexpression) would directly connect chromatin binding to the regulation of fate determinants. These investigations would strengthen the mechanistic connection between the correlative TaDa profiles and the observed identity changes, supporting the idea that Gsb functions as a context-dependent chromatin-priming factor within the STF code, rather than as a generic transcription factor.
We thank the reviewer for this suggestion. We agree that examining Gsb occupancy at known lineage determinants helps connect the genome-wide TaDa profiles to the developmental phenotypes. We have therefore added browser profiles showing endogenous Gsb occupancy at several loci associated with NB5-6 identity and its neuronal output, including lbe, ap, and eya (new Figure 2E). These are the same markers that are lost following Gsb depletion and partially gained following Gsb misexpression (Figures 7 and 8), providing a locus-level connection between Gsb binding and the observed changes in NB5-6 identity.
The precise comparison suggested by the reviewer is difficult because the binding and misexpression experiments were necessarily performed in different contexts. Gsb occupancy was measured in NB5-6, where Gsb is normally expressed, whereas the effect of Gsb misexpression on chromatin was measured in the heterologous NB7-4 context where Gsb is not normally expressed. We therefore did not measure Gsb occupancy following its misexpression in NB7-4 and so cannot compare Gsb binding before and after misexpression at individual loci in NB7-4.
We have nevertheless added genome-browser examples showing how Gsb misexpression affects accessibility and Hb occupancy at lineage-associated loci (new Figure 6M). The ems locus provides one such example: ectopic Gsb reduces both accessibility and Hb occupancy at ems in NB7-4, while broad Gsb misexpression causes loss of Ems expression and of the independent NB3-5 reporter R59E09>GFP in the phenotypic experiments (Figure 8).
(2) Gsb misexpression reveals bidirectional chromatin remodelling
Experiments with ectopic Gsb expression demonstrate bidirectional chromatin remodelling in NB7-4, showing decreases in accessibility at some binding sites and increases at others. While the authors show that Gsb can disrupt chromatin upon misexpression, interpreting its "pioneer-like" or chromatin-priming activity is complex due to several factors: the misexpression occurs in a non-native lineage, the direct versus indirect effects rely on whole-embryo Dam-Gsb peaks instead of NB7-4-specific binding, and heat-shock-induced chromatin changes are not fully accounted for. These issues make it challenging to definitively determine Gsb's role in chromatin priming.
A complementary approach would be to perform Gsb knockdown/loss-of-function in its native NB5-6 lineage and profile chromatin accessibility (TaDa or CATaDa). This would allow a cleaner, more physiologically relevant assessment of Gsb's contribution to priming, SoI establishment, and Hb recruitment. Such an experiment would strengthen the causal link between Gsb occupancy and chromatin state and clarify whether Gsb truly acts as a context-dependent pioneer in vivo, rather than producing indirect effects due to ectopic misexpression.
We thank the reviewer for this thoughtful comment. The reviewer raises four related but distinct issues, which we address separately below.
(1) Gsb was misexpressed in a non-native lineage
As discussed in our response to Reviewer 1 regarding pioneer activity, expression in a heterologous cellular context is a standard experimental strategy for testing whether a transcription factor has the capacity to engage and remodel chromatin outside its endogenous setting. The use of NB7-4 was therefore deliberate: this experiment tested the sufficiency of Gsb to alter chromatin, rather than attempting to reproduce its physiological activity in NB5-6. Nevertheless, because Gsb produced bidirectional changes rather than autonomous chromatin opening, we have revised our interpretation. We now describe this experiment as demonstrating chromatin remodelling capacity and use “chromatin priming” rather than claiming a classical pioneer for its activity.
Relevant edits made to the manuscript: Results, Figure 7 and Figure 7—figure supplements 1 and 2.
(2) Direct and indirect effects were inferred using whole-embryo Gsb occupancy
We have not profiled Gsb occupancy directly in NB7-4 following its misexpression. However, we have now performed an unbiased genome-wide analysis using two independent Gsb-binding datasets: stage-matched Gsb occupancy from NB5-6 and whole-embryo Gsb occupancy. Since Dam methylation is cumulative across the window of Gal4 activity, the whole-embryo dataset serves as a proxy for embryowide sites that are compatible with Gsb occupancy. Across both datasets, the result was strikingly consistent. The set of 1,340 loci that lost accessibility upon Gsb misexpression showed strong Gsb occupancy in both datasets, whereas the 606 loci that gained accessibility showed little or no Gsb occupancy in either dataset (Figure 6E,H). Thus, two independently generated Gsb-binding maps systematically distinguish the loci that close from those that open.
This reproducibility — across many sites and two datasets — provides strong evidence that accessibility loss occurs at loci that Gsb can bind and is therefore likely to reflect a direct consequence of Gsb activity. Conversely, lower Gsb occupancy at sites that gain accessibility suggests that these gains are more likely to be indirect. The corresponding changes in Hb occupancy further strengthen this interpretation (Figure 6C–G).
In summary, we find it particularly compelling that two independently generated Gsb-binding maps, obtained at different developmental stages and in different cellular contexts, identify the same systematic distinction: loci that close upon Gsb misexpression are Gsb-bound, whereas loci that open are not. We therefore consider this a strong and unbiased result supporting a direct role for Gsb in chromatin closing.
Relevant edits made to the manuscript: (Results; Figure 6E, H)
“Although we did not directly profile Gsb occupancy in NB7-4 upon misexpression, we asked whether the sites that lose or gain accessibility differ in their propensity to be bound by Gsb. To do this, we mapped independent Gsb occupancy datasets onto these two classes of remodelled sites: stage-matched Dam: Gsb from NB5-6, where Gsb is normally expressed, and whole-embryo Dam: Gsb, which provides a broader map of Gsb-compatible loci. In both datasets, sites that lost accessibility upon Gsb misexpression in NB7-4 showed strong Gsb occupancy, whereas sites that gained accessibility showed little or no Gsb signal. Thus, the most direct consequence of ectopic Gsb is likely accessibility loss at loci that Gsb can bind, and this loss is accompanied by reduced Hb occupancy. By contrast, accessibility gains occur mainly at loci with little evidence of Gsb binding in independent datasets, suggesting that these gains are more likely indirect consequences of Gsb misexpression.”
(3) Heat-shock-induced chromatin changes were not fully accounted for
We agree that, because heat shock is required to induce Gsb, a contribution from the induction regime cannot be completely excluded. However, several observations argue that the chromatin changes are not dominated by a non-specific heat-shock response. First, the accessibility changes were reproducible across biological replicates (Figure 6—figure supplement 2). Second, as described above, sites that lost accessibility were consistently occupied by Gsb in two independent datasets and showed a corresponding reduction in Hb occupancy, whereas sites that gained accessibility showed little or no Gsb occupancy (Figure 6C–H). This structured relationship would not be readily explained by a general heat-shock response.
As an additional test, we performed a Gene Ontology analysis of genes associated with sites that gained accessibility. The enriched terms were predominantly related to development and cell-fate specification, including neuroblast differentiation and segment specification, while heat-shock and stress-response terms were not enriched (Author response image 1). Although none of these observations alone formally excludes an effect of heat shock, together they argue that the observed pattern reflects structured, Gsb-associated chromatin remodelling rather than a general stress response. We nevertheless interpret the accessibility gains cautiously as likely indirect effects of Gsb misexpression.
Author response image 1.
Gene ontology analysis of sites showing gain in accessibility upon Gsb misexpression in NB7-4. Genes overlapping the 606 sites showing at least 2-fold increase in accessibility (DamOnly signal) were annotated using the UCSC Table Browser (dm6). The resulting gene list was analysed for associated gene ontology (GO) terms using Metascape (Zhou et al. 2019). The top 20 enriched GO terms are represented as a bar chart, coloured by p-values.
(4) Chromatin accessibility following Gsb loss in NB5-6 would provide a more physiological test
We agree that this would be an informative experiment, and we explored several ways of perturbing Gsb early enough to address its endogenous function. A NB5-6-specific RNAi approach could not be used because the available NB5-6 Gal4 driver becomes active only after the period in which Gsb specifies the lineage.
The difficulty with conventional gsb mutants is different but equally limiting. Homozygous embryos would have to be identified during the brief developmental window in which Hb is expressed, while each TaDa/CATaDa replicate requires at least 30 mg of precisely staged embryos. The available fluorescent balancer markers are either not detectable at these early stages or become reliably detectable only towards the end of the window. It is therefore not feasible to identify and collect sufficient homozygous embryos — either manually or by fluorescence-assisted whole-embryo sorting — within the required time frame.
We therefore generated a gsb guide-RNA line and used Vasa-Cas9 to deplete Gsb in the early embryo. This strategy produced a high rate of Gsb editing, embryonic lethality and the expected axonal defects (Figure 7—figure supplement 1). When we examined NB5-6 in these embryos, we found that the Lbe-K reporter and the NB5-6 neuronal markers Ap and Eya were lost, confirming the requirement for Gsb in specifying this lineage (Figure 7).
Unfortunately, this also meant that we could not take the experiment forward for TaDa/CATaDa. Lbe-K-Gal4 is the driver we use to target NB5-6, and its expression was lost following early Gsb depletion. Without the driver, we can no longer restrict Dam expression to NB5-6 and therefore cannot obtain a lineage-specific chromatin profile. Thus, although the early CRISPR strategy allowed us to examine the developmental consequences of Gsb loss, the same experiment could not be used to assay chromatin accessibility in NB5-6. We have made this limitation explicit in the revised Discussion.
Relevant edits made to the manuscript: (Result, Figure 7 and Figure 7— figure supplement 1). We also now state explicitly in the “Scope and future directions” section that the current tools did not allow us to profile the chromatin consequences of Gsb loss directly in NB5-6 (Discussion).
(3) En is not a pioneer factor
The authors conclude that Engrailed (En) is not a pioneer factor, based on the observation that En binding correlates with accessible chromatin and that En is not enriched at NB5-6-specific SOIs. However, this conclusion is not sufficiently supported by the functional data.
First, the absence of En binding at NB5-6-specific SOIs does not necessarily indicate an inability to engage closed chromatin. These regions were not selected for the presence of En consensus motifs, so their lack of occupancy may simply reflect the absence of En binding motifs rather than a lack of pioneering capacity. A systematic motif analysis at NB5-6-specific SOIs is needed to determine whether En binding sites are present but unoccupied.
Second, the claim that En lacks pioneer activity relies solely on a single steady-state TaDa/DamID occupancy assay at one developmental stage. Because pioneer factor interactions can be transient, low-affinity, and stage-specific, such binding may not be detected by TaDa, which also depends on local GATC density and methylation kinetics and may yield false negatives. Given these technical limitations, the absence of En binding at less accessible regions does not definitively rule out a priming role.
In the absence of direct functional assays (En LOF/GOF), the authors should explicitly acknowledge these technical and conceptual limitations and tone down the claim that "En lacks pioneer activity".
We thank the reviewer for raising this point. We agree that the statement “En is not a pioneer factor” could be read as a general conclusion about En activity across developmental contexts. Our conclusion is more specific: in NB7-4 during the Hb-competence window, En occupancy is strongly biased towards pre-accessible chromatin and does not display the chromatin-engagement pattern expected of a pioneer factor. We have revised the manuscript throughout to make this context-specific interpretation clear. Below, we also address each of the reviewer’s points.
(1) The absence of En at NB5-6-specific SoIs could reflect an absence of En-binding motifs
We agree that the absence of En occupancy at NB5-6 SoIs would not be informative if these loci lacked En-binding motifs. We therefore performed the suggested motif analysis. En motifs were present at comparable frequencies in NB5-6- and NB7-4-specific SoIs (88.0% and 86.5%, respectively), with both enriched relative to matched shuffled controls (new Figure 3—figure supplement 1L). Thus, NB5-6 SoIs are motif-compatible but remain unoccupied by En in NB7-4. Their lack of En occupancy cannot therefore be explained simply by motif absence and instead points to additional determinants such as chromatin accessibility or cofactor context.
Relevant edits made to the manuscript: Result, Figure 3B–C and new Figure 3—figure supplement 1L)
“As expected, En was enriched at NB7-4-specific SoIs, where Hb uniquely binds in this lineage. However, there was no detectable En signal in NB7-4 at sites known to be NB5-6-specific SoIs, which remain relatively less accessible and Hb-free in NB7-4. This difference is unlikely to be explained simply by En motif availability, since En motifs are similarly represented at NB7-4- and NB5-6-specific SoIs. Thus, at SoIs, En occupancy is associated with the cognate, accessible NB7-4 SoIs, with little detectable occupancy at the non-cognate, relatively less-accessible NB5-6 SoIs.”
(2) TaDa may fail to detect transient, low-affinity or stage-specific En binding
We agree that no occupancy assay can exclude interactions below its detection threshold. However, TaDa is cumulative rather than a steady-state snapshot: Dam methylation accumulates during the expression window, allowing transient interactions to leave a detectable signal. Genome-wide, En occupancy was strongly weighted towards accessible chromatin, and accessibility predicted En binding with an AUROC of 0.74 (Figure 3E–I, Figure 3—figure supplement 2 and new Figure 3— figure supplement 3E).
Gsb provides an internal comparison using the same experimental framework. Gsb binding was detected at both accessible and relatively less-accessible chromatin, and accessibility did not predict Gsb occupancy better than chance (AUROC = 0.41; Figure 4E–I and new Figure 3—figure supplement 3E). This argues that the En pattern is not caused by a general inability of TaDa to detect binding at less-accessible loci. We nevertheless acknowledge that rare interactions, interactions below the detection threshold or binding outside the assayed window cannot be excluded. The resolution and cumulative nature of TaDa are now also described explicitly in the revised manuscript.
Relevant edits made to the manuscript: Result section for the genome-wide analysis and Discussion section for the TaDa limitations; Figure 3E–I, Figure 4E–I, Figure 3—figure supplement 2 and new Figure 3—figure supplement 3E).
(3) Direct functional assays of En were not performed
We agree that determining whether En actively establishes or maintains chromatin accessibility would require En loss- or gain-of-function experiments combined with chromatin profiling. These experiments were not performed in this study, and we have therefore removed the categorical statement that “En is not a pioneer factor.”
The revised manuscript now states the more specific conclusion supported by our data: En occupancy in NB7-4 during the Hb-competence window is strongly biased towards pre-accessible chromatin. We also acknowledge that the current experiments cannot exclude rare or transient interactions below the detection threshold, or chromatin-remodelling activity by En at other loci, developmental stages or cellular contexts. The factors that establish the NB7-4 accessibility landscape therefore remain unresolved.
Relevant edits made to the manuscript: (Result; Figure 3)
“Taken together, these results show that En occupancy is strongly biased toward preaccessible chromatin in NB7-4, including at NB7-4-specific SoIs. Thus, En is well placed to integrate spatial and temporal information through direct regulation at preaccessible enhancers. However, because NB7-4 has a distinct accessibility landscape, the factors that establish this landscape remain unresolved. We therefore propose that NB7-4 may use a combinatorial STF-code logic, in which other lineage-specific factors help establish NB7-4-specific SoIs, while En occupies this pre-accessible landscape and likely collaborates with Hb to activate lineage-specific enhancers.”
(4) Clarity of STF-code Model and Central Message
The manuscript begins by presenting two models, direct and epigenetic, but the central takeaway of the paper is not clear. Specifically, the nuanced roles of the spatial factors Gsb and En as chromatin-priming versus stabilizing/effector factors within an STF code, and the resulting division of labor, are not clearly illustrated. The distinction between Gsb as a chromatin-priming factor and En as a cofactor-dependent activator/stabilizer should be explicitly presented in a stepwise model for better clarity. The authors could strengthen this by providing a schematic with two sequential stages illustrating how neuroblast identity factors (STF code) change chromatin states to drive lineage-specific enhancer activation. The schematic can be shown from the neuroectoderm to individual NB lineages to make it more panoramic.
We thank the reviewer for this suggestion. This comment, together with related points raised below, made us realise that the manuscript did not bring out strongly enough that our data lead us to propose a third model: the distributed STF-code model. Rather than treating direct and epigenetic regulation as mutually exclusive alternatives, this model distributes these activities across different members of a NB-specific STF code. We have now revised the manuscript to reflect this transition. For example, we now state this explicitly in the Abstract, at the end of the Introduction and at the beginning of the Discussion.
Relevant edits made to the manuscript: Abstract, Introduction, Discussion, and have added a new summary schematic (new Figure 9).
(5) Identification of Priming Factors in NB7-4
While the authors suggest that an unknown priming factor might be responsible for establishing sites of integration in NB7-4, they do not identify or explore potential candidates for this role. Further investigation into what factors might be involved in chromatin priming in NB7-4 could provide a more complete understanding of the mechanisms at play.
We thank the reviewer for this suggestion. We agree that identifying the factors that establish the NB7-4 accessibility landscape is an important next step. Our current data do not identify such a factor, and doing so would require systematic perturbation of candidate STF-code members together with lineage-specific chromatin profiling.
We have nevertheless made this question more concrete in the revised Discussion. We identify Msh as a candidate shared input in NB5-6 and NB7-4, highlight additional candidates suggested by motif enrichment at NB7-4 SoIs, and propose NB6-4 — which coexpresses Gsb and En — as a useful context in which to test the division of labour between STF-code members. We present these as testable candidates rather than established priming factors.
(6) Functional Validation of STF Code Components
The study proposes an STF code for each neuroblast lineage, but the specific components of these codes, beyond Gsb and En, are not fully explored. Identifying and validating additional factors that contribute to the STF code in each lineage could strengthen the conclusions.
We thank the reviewer for this point. We agree that the complete composition of the NB5-6 and NB7-4 STF codes remains to be established, and we now make this explicit. The present study proposes a mechanistic framework based on the different chromatin-engagement properties of Gsb and En, rather than claiming to have defined or validated every component of either code.
As noted in our response above, the revised Discussion identifies Msh, Iroquois-complex members and TF families enriched at NB7-4 SoIs as candidate additional components. Testing these factors individually and in combination, with chromatin, Hb-occupancy and lineage-identity readouts, will be required to define the minimal functional code for each lineage. We present this as an important direction for future work (lines 759–770).
Recommendations for the authors:
Reviewer #1 (Recommendations for the authors):
(1) The reviewers appreciate the content shown in Figure 1, which is helpful for people who are unfamiliar with the system as a whole. One key issue is that later in the manuscript, the authors look at Gsb and describe it as a factor that works to close chromatin, making the model depicted in panel E misleading when reading the first half of the manuscript and leading to confusion/whiplash when getting to Figure 6, where a spatial transcription factor is thought to play a role in closing chromatin.
Thank you for pointing this out. Figure 1 was intended to present the two models currently proposed in the field and as the starting hypotheses for the study. It was not intended to present the conclusions from our study. We realise, however that this distinction was not clear and since we did not present the updated model, it made the later finding appear contradictory.
We have now made substantial edits to the text and have made the transition from the original models to our revised interpretation explicit in the Introduction and Discussion. We have also added a new summary model (new Figure 9), which shows that chromatin opening, chromatin closing and STF–Hb engagement can be distributed across different members of an NB-specific STF code.
(2) It is unclear what "less accessible" actually means. The reviewers would like to see quantitative standardization to define the degree of chromatin accessibility when using the Dam expression alone.
We thank the reviewer for this point. The comment made us revisit how we defined accessibility, which we had previously treated too much as a binary distinction between accessible and inaccessible chromatin. We have now revised our analyses to represent chromatin accessibility as a continuum of states.
We defined a common set of 27,119 DamOnly-marked regions across both NBs, ranked them by mean DamOnly signal and divided them into deciles from least to most accessible. The construction and validation of this continuum are shown in new Figure 2G and new Figure 2—figure supplement 2A–D. We also use this framework to examine STF occupancy and Gsb-dependent chromatin remodelling across the accessibility spectrum (new Figure 3— figure supplement 2A and new Figure 6—figure supplement 2E–F).
Where a discrete lineage-specific comparison was required, we applied a minimum two-fold difference in DamOnly signal between the NBs. This identified 845 NB5-6-preferential and 1,067 NB7-4-preferential regions. A region preferentially accessible in one NB is therefore described as relatively less accessible in the other, rather than as completely inaccessible.
Relevant edits made to the manuscript: Results, new Figure 2G and new Figure 2—figure supplement 2A–D.
(3) A key takeaway in Figure 3 is that En is binding already accessible chromatin. Another key point that this manuscript makes is that spatial-temporal factors (and codes) are responsible for this opening. We have not yet been shown that chromatin accessibility of this neuroblast identity actually changes upon spatial-temporal factor expression. I suggest including data of the dam-only control as a time course. Showing chromatin accessibility just before temporal factor expression and then after to validate that pioneer factor activity appears to be likely.
We appreciate this suggestion and agree that, conceptually, the model implies a temporal sequence in which spatial factors first act during neuroectoderm patterning and temporal factors such as Hb subsequently engage the chromatin after neuroblast formation. In practice, however, these events occur in very rapid succession during development. Spatial transcription factors are expressed in the neuroectoderm, neuroblasts are specified and delaminate, and Hb expression begins almost immediately after neuroblast formation — all within a short developmental window on the order of minutes to tens of minutes.
This makes it difficult to directly test chromatin accessibility changes across these stages using lineage-restricted Dam-based approaches. Before delamination there is no neuroblast to target with the available drivers, and once the neuroblast forms the temporal program is already underway. As a result, the sequential changes in accessibility that the reviewer is suggesting cannot currently be resolved with the available tools.
We agree that directly visualizing how chromatin accessibility changes during this brief developmental transition would be very interesting, but doing so would likely require approaches with much higher temporal resolution, such as live imaging-based methods for monitoring chromatin accessibility.
(4) Similar to the comment for Figure 3, showing chromatin accessibility changes in the NB subtype via a time course that includes both before and after spatial transcription factor expression begins.
This point is closely related to the reviewer’s previous comment regarding a temporal analysis of chromatin accessibility. Please refer to our response above describing the developmental timing and technical limitations that make such an experiment difficult in this system.
(5) It is difficult to reconcile the conclusion in Figure 5 that regions bound by both En/Hb or Gsb/Hb are considered the highest accessibility. Can the authors touch on the logic for this, given that in the next figure, they are going to suggest that Gsb works as a factor that is primarily responsible for closing chromatin? It is unclear how Gsb is closing chromatin, but it is also binding Hb, and in those regions, chromatin is most accessible.
We thank the reviewer for raising this point. The findings in Figures 5 and 6 may initially appear difficult to reconcile, but we think that they both actually point to the same conclusion: that Gsb’s effect on chromatin is context-dependent.
An important point is that Gsb binding is not restricted to a single chromatin state. In its endogenous NB5-6 context, it occupies both accessible and relatively less-accessible regions (Figure 4B–C, E–F). Its presence at accessible chromatin does not establish that Gsb opened that chromatin nor that it closed it because these occupancy data do not reveal the direction of chromatin change. From these data we can only conclude that sites co-bound by Gsb and Hb are more accessible than sites bound by Gsb alone, suggesting that the outcome of Gsb binding depends on the surrounding regulatory context. In the presence of Hb — and likely other members of the NB5-6 STF code — Gsb can participate in a highly accessible, productive regulatory state (Figure 5C–E).
The misexpression experiment helps us interpret these relationships in the endogenous context. When Gsb is introduced into NB7-4, sites that lose accessibility are strongly Gsb-bound in two independent datasets, and this loss is accompanied by reduced Hb occupancy (Figure 6C–H). Some Gsb-bound sites also gain accessibility, consistent with bidirectional remodelling, but Gsb occupancy is negligible at these gain sites. Thus, outside its normal NB5-6 regulatory environment, the more direct effect of Gsb binding is likely chromatin closing.
This provides a possible explanation for the endogenous (NB5-6) data in Figure 5. The lower accessibility of sites bound by Gsb alone may reflect this closure-biased activity, whereas the highly accessible state at Gsb–Hb co-bound sites is likely to arise from the broader NB5-6 regulatory context. We do not conclude that every Gsb-only site is actively closed, or that Gsb itself opens the co-bound sites. Rather, our data suggest that Gsb can participate in different chromatin states, with the outcome of its binding shaped by Hb and other members of the STF code.
We have revised the Results and Discussion to make this interpretation clearer.
(6) The most striking piece of data from Figure 6 is that it appears the physiological role of Gsb is to reduce chromatin accessibility and that opening chromatin is an indirect effect. I am not sure that the field widely recognizes closing as chromatin as "pioneer-like" activity. Moreover, this finding seemed very out of place given the way the story has been sold thus far - discussing "priming of chromatin" and referring to this as opening in figure 1 and discussion of binding of these factors at open and less accessible.
Thank you for raising this issue, which overlaps with the reviewer’s previous point. We were equally struck by the result from Figure 6: the sites that lose accessibility are more consistent with a direct effect of Gsb, whereas the sites that gain accessibility are more likely to represent indirect effects. This suggests that Gsb can remodel chromatin bidirectionally.
We agree that the field does not generally describe chromatin closing as pioneer activity. We therefore no longer refer to Gsb as a pioneer factor. Instead, we describe Gsb as having bidirectional chromatin-remodelling capacity and refer to it as “chromatin priming” activity.
Finally, we agree that the original presentation made this progression difficult to follow. Figure 1 presents the two existing models as the starting point, whereas our data lead us to propose a third, distributed STF-code model. We now make this transition explicit throughout the revised manuscript and have added a new summary schematic (new Figure 9; See Results and Discussion).
Reviewer #2 (Recommendations for the authors):
Minor comment:
(1) Have the authors validated the specificity of the Lbe-GAL4 and 19B03AD;18F07DBD drivers? It is important to clarify whether these drivers label only NB5-6 and NB7-4 neuroblasts or also their progeny. Since the interpretation of chromatin dynamics is in the context of neuroblasts, reads from differentiated neurons could affect the conclusions.
We thank the reviewer for raising this point. These driver lines were extensively characterised in our previous study and are highly specific to the NB5-6 and NB7-4 lineages, respectively. Importantly, as the reviewer points out, they are not neuroblast-exclusive and also label progeny. Because our profiling is restricted to the early Hb competence window, this contribution should be limited to a small number of GMCs and the earliest-born neurons.
On rereading the manuscript, we realised that there were several instances where we used “NB-specific” too loosely when referring to these datasets. We have therefore revised the manuscript throughout to make clear that the TaDa/CATaDa profiles represent the NB5-6 and NB7-4 lineages during the Hb window and are not exclusively derived from the neuroblast itself. We have also added this explicitly as a limitation in the Scope and Future Directions section. Importantly, this does not change the essence of our conclusions, which are based on comparisons between these two defined lineages and on perturbations examined within the same lineage-restricted framework.
Relevant edits made to the manuscript:
- “The driver lines were previously validated as being specific to these lineages”.
- “First, the lineage-specific Gal4 drivers also label the immediate progeny of each NB; thus, although profiling was restricted to the early Hb competence window, when only a small number of GMCs and early-born neurons are present, the resulting profiles are not exclusively derived from the NB itself.”
(2) The lineage NB6-4 co-expresses both Gsb and En and represents an informative intermediate case. This context supports a cooperative STF-code model in which Gsb provides chromatin priming while En acts as an effector or stabilizer, without requiring autonomous pioneer activity. The authors should briefly discuss how NB6-4 fits into their model and whether Gsb priming in this lineage enables En-Hb co-occupancy and enhancer activation.
We thank the reviewer for this comment. We agree that NB6-4 is an especially interesting case in the context of our model, because it co-expresses both Gsb and En and therefore may provide a lineage in which both aspects of the proposed two-step mechanism can be examined together: Gsb could contribute to chromatin priming and En could function at already primed enhancers together with Hb. We now include this explicit example in the Discussion section.
(3) The authors' data support a role for Gsb in lineage-specific chromatin priming, but the functional experiments reveal important limitations. Gsb alone does not efficiently open closed chromatin, install NB5-6-specific SOIs, or recruit Hb, and ectopic Gsb often antagonizes native chromatin states. Thus, the results do not support Gsb as a classical standalone pioneer factor, as implied in the discussion. The authors should replace the term "pioneer-like potential or activity" with "context-dependent pioneer activity" in the results, as they have rightly mentioned in the discussion.
We agree with the reviewer. As discussed in our responses to the related comments above, our data do not support describing Gsb as a classical pioneer factor. Gsb remodels chromatin bidirectionally, with both gains and losses of accessibility, whereas pioneer activity is traditionally associated with chromatin opening. We have therefore gone further than the reviewer’s suggested change: throughout the manuscript, we have replaced “pioneer-like potential/activity” and “context-dependent pioneer activity” with “context-dependent chromatin remodelling” or “chromatin priming.” The Results and Discussion now make it clear that Gsb’s activity depends on the surrounding STF and cofactor context rather than representing autonomous pioneer activity.
(4) The use of ectopic expression systems may introduce artifacts or off-target effects that could affect the interpretation of the results. These potential limitations are not extensively discussed.
We agree that ectopic expression can produce indirect or non-physiological effects. The Gsb misexpression experiment was designed to test whether Gsb is sufficient to remodel chromatin in the heterologous NB7-4 context, rather than to reproduce its endogenous function in NB5-6. We therefore do not interpret every change in accessibility in the misexpression experiment as a direct or physiological role for Gsb in NB5-6. However, the changes are not random. Sites that lose accessibility are strongly associated with Gsb occupancy in two independent datasets and they also lose Hb occupancy, whereas sites that gain accessibility show little Gsb occupancy and are therefore more likely to reflect indirect effects (Figure 6C–H). The changes were reproducible between replicates, and genes associated with accessibility gains were not enriched for heat-shock or stress-response terms (Author response image 1). These analyses do not remove the limitations of ectopic expression, but they help distinguish the more likely direct effects from indirect ones. We now make both the purpose and the limitations of this experiment explicit in the Results and Discussion.
(5) The moderate correlation (0.7-0.82) between Dam-En and Dam-only reflects the expected overlap of En binding with accessible chromatin, but it also underscores a limitation: TFs that prefer open regions may show high baseline similarity to Dam-only, potentially masking low-affinity or transient interactions. The authors should acknowledge this when concluding that En lacks pioneer activity.
We thank the reviewer for bringing this up. We want to clarify that our conclusion that En preferentially occupies accessible chromatin is not based only on the moderate genome-wide correlation between Dam: En and DamOnly. We also examined En binding at defined SoIs and at differentially accessible loci across the genome. In both analyses, En is strongly enriched at accessible NB7-4 sites but shows little detectable enrichment at the corresponding less-accessible sites (Figure 3B–F). We have now extended this analysis across 27,119 regions ranked along the full accessibility spectrum. Here too, En signal remains low across the less-accessible bins and increases principally in the most accessible bins (new Figure 3— figure supplement 2E).
A useful comparison here is Gsb. Using the same experimental and analytical framework, we readily detect Gsb binding at both accessible and relatively less-accessible chromatin in NB56 (Figure 4B–F; new Figure 3—figure supplement 2E). Thus, the method is able to detect binding at less-accessible chromatin when it occurs. Consistent with this, accessibility predicts En occupancy but not Gsb occupancy in the corresponding ROC analysis (new Figure 3—figure supplement 3E). We also refer the reviewer to our response to the related comment above, where we discuss this comparison in more detail.
Finally, we want to point out that TaDa is not a snapshot assay. Dam-mediated methylation accumulates over the expression window and across many cells, so repeated weak or transient interactions should, in principle, contribute to the signal. We agree, however, that very rare interactions below the detection threshold cannot be excluded. We have therefore tempered our conclusion throughout the manuscript: rather than stating that En lacks pioneer activity, we now conclude that En occupancy during the NB7-4 Hb window is strongly biased towards pre-accessible chromatin.
(6) The schematic illustrating the NB 5-6 and NB 7-4 SOIs (Figures 3A, 4A, and S2A) should be made clearer. When Hb is bound, sites that are accessible serve as SOIs for a specific lineage, whereas Hb-unbound, inaccessible sites do not. The authors can visually represent this by showing that, in the case of Hb binding, the accessible region functions as a resident SOI for the NB7-4 lineage, while the same region in NB5-6, being Hb-free, is inaccessible. This distinction could be indicated with a shaded or shadowed area from top to bottom in Figures 3A and 4A.
We thank the reviewer for pointing this out. We have revised these panels to make the visual distinction between lineage-specific SoIs and the corresponding Hb-free, less-accessible loci in the other lineage more explicit.
(7) At 472, change "Fig.S6C-F" to "Fig.S6C-D".
Thank you for spotting that!
